Benzylideneguanidine derivatives and therapeutic use for the treatment of protein misfolding diseases

ABSTRACT

The present invention relates to a compound of formula (I), or a tautomer and/or a pharmaceutically acceptable salt thereof, wherein R 1  is alkyl, Cl, F or Br, R 2  is H or F; R 3  is selected from H and alkyl; R 4  is selected from H and C(O)R 6 ; R 5  is H or R 4  and R 5  are linked to form a heterocyclic group which is optionally substituted with one or more R 10  groups R 6  is selected from R 7 , OR 7  and NR 8 R 6 ; R 7 R 8  and R 9  are each independently selected from alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocycyl and aryl, each of which is optionally substituted with one or more R 10  groups; each R 10  is independently selected from halogen, OH, CN NO 1 , COO-alkyl, aralkyl, SO 2 -alkyl, SO 2 -aryl, COOH, CO-alkyl, CO-aryl, NH 2 , NH-alkyl, N(alkyl) 2 , CF 3 , alkyl and alkoxy, X and Z are each independently CR 11  and Y is selected from CR 11  and N and R 11  is H or F; for use in treating a disorder associated with protein misfolding stress and in particular associated with accumulation of misfolded proteins.

This application is a continuation of U.S. application Ser. No.15/467,613, filed Mar. 23, 2017, which is a continuation of U.S.application Ser. No. 14/760,350, filed Jul. 10, 2015, now U.S. Pat. No.9,682,943, issued Jun. 20, 2017, which is a U.S. national phaseapplication under 35 USC 371 of International Patent Application no.PCT/EP2014/050422, filed Jan. 10, 2014, which claims the benefit ofGreat Britain Application no. 1300435.3, filed Jan. 10, 2013.

The present invention relates to compounds that have potentialtherapeutic applications in treating disorders associated with proteinmisfolding stress and in particular with an accumulation of misfoldedproteins. In particular, the invention provides compounds that arecapable of exhibiting a protective effect against cytotoxic endoplasmicreticulum (ER) stress.

BACKGROUND TO THE INVENTION

The compound 2-(2,6-dichlorobenzylidene)hydrazinecarboximidamide, alsoreferred to as guanabenz, is an alpha agonist of the alpha-2 type thatis used as an antihypertensive drug.

Various derivatives of guanabenz have also been reported. For example,U.S. Pat. No. 3,982,020 (Sandoz, Inc.) discloses substituted benzylidenehydrazines and their use as hypoglycemic-antihyperglycemic agents,anti-obesity agents and anti-inflammatory agents. US 2004/0068017(Bausch & Lomb Inc.) discloses substituted benzylidene hydrazines thatare capable of increasing the activity of gelatinase A in ocular cells.The molecules have applications in the treatment of primary open angleglaucoma. WO 2008/061647 (Acure Pharma AB) discloses the use ofN-(2-chloro-3,4,-dimethoxybenzylideneamino)guanidine as a VEGFRinhibitor and its associated applications in the treatment or preventionof undesired blood vessel formation during tumour growth and/orinflammatory conditions. WO 2005/031000 (Acadia Pharmaceuticals, Inc.)discloses substituted benzylidene hydrazines and their use in treatingacute pain and chronic neuropathic pain. Finally, EP 1908464 (CNRS)discloses guanabenz and chloroguanabenz and their use in the treatmentof polyglutamine expansion associated diseases, including Huntington'sdisease.

More recently it has been reported that guanabenz has therapeuticpotential in a number of other areas. Guanabenz, was recently noted tohave anti-prion activity (D. Trobouillard-Tanvier et al., 2008 PLoS One3, e1981). It has been reported that its activity in protecting againstprotein misfolding is surprisingly much broader and includes attenuatingaccumulation of mutant Huntingtin in cell-based assays (WO 2008/041133)and protection against the lethal effects of expression of misfoldingprone Insulin Akita mutant in the endoplasmic reticulum (ER) of Min6 andINS-1 pancreatic beta-cells (P. Tsaytler, H. P. Harding. D. Ron and A.Bertolotti, Science, 332, 1 Apr. 2011, 91-94).

Guanabenz has also been shown to promote survival of HeLa cells exposedto otherwise cytotoxic ER-stress induced by the N-glycosylationinhibitor tunicamycin, in a dose-dependent manner (P. Tsaytler, H. P.Harding. D. Ron and A. Bertolotti, Science, 332, 1 Apr. 2011, 91-94).Quantitative assessment of cell viability revealed that guanabenzdoubled the number of cells surviving ER stress with a median effectiveconcentration of ˜0.4 μM. Neither the α2-adrenergic receptor agonistclonidine, nor the α2-adrenergic receptor antagonist efaroxan protectedcells from cytotoxic ER stress and efaroxan did not interfere withguanabenz's protective effect (P. Tsaytler, H. P. Harding, D. Ron and A.Bertolotti, Science, 332, 1 Apr. 2011, 91-94). These observationsdemonstrate that guanabenz rescues cells from lethal ER stress by amechanism independent of the α2-adrenergic receptor. Guanabenz protectscells from otherwise lethal accumulation of misfolded proteins bybinding to a regulatory subunit of protein phosphatase 1, PPP1R15A(GADD34), selectively disrupting the stress-induced dephosphorylation ofthe α subunit of translation initiation factor 2 (elF2α). Guanabenz setsthe translation rates in stressed cells to a level manageable byavailable chaperones, thereby restoring protein homeostasis. It wasreported that Guanabenz does not bind to the constitutive PPP1R15B(CReP) and therefore does not inhibit translation in non-stressed cells.(P. Tsaytler, H. P. Harding, D. Ron and A. Bertolotti, Science, 332, 1Apr. 2011, 91-94).

Failure to maintain proteostasis in the ER by mounting an adequateunfolded protein response (UPR) is recognized as a contributing factorto many pathological conditions. Thus, the molecules described here,which inhibit elF2α phosphatase to fine-tune protein synthesis, may beof therapeutic benefit to a large number of diseases caused proteinmisfolding stress and in particular with an accumulation of misfoldedproteins.

The present invention seeks to provide alternative compounds based on aguanabenz core structure that have potential therapeutic applications intreating disorders associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins.

STATEMENT OF INVENTION

A first aspect of the invention relates to a compound of formula (I), ora pharmaceutically acceptable salt thereof,

wherein:R₁ is alkyl, Cl, F or Br;R₂ is H or F;R₃ is selected from H and alkyl;R₄ is selected from H and C(O)R₆;R₅ is H;or R₄ and R₅ are linked to form a heterocyclic group which is optionallysubstituted with one or more R₁₀ groups;R₆ is selected from R₇, OR₇ and NR₈R₉;R₇, R₈ and R₉ are each independently selected from alkyl, cycloalkyl,aralkyl, cycloalkenyl, heterocyclyl and aryl, each of which isoptionally substituted with one or more R₁₀ groups;each R₁₀ is independently selected from halogen, OH, CN, COO-alkyl,aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl,N(alkyl)₂, CF₃, alkyl and alkoxy;X and Z are each independently CR₁₁, and Y is selected from CR₁₁ and N;R₁₁ is H or F;for use in treating a disorder associated with protein misfolding stressand in particular with an accumulation of misfolded proteins.

Previous studies have indicated that the aryl group must be at leastdisubstituted in order for the compounds to exhibit usefulpharmacological activity (see for example, D. Tribouillard-Tanvier etal., PLoS One 3, e1981 (2008) and EP1908464A, CNRS). However, contraryto the results of previous studies, the present Applicant hassurprisingly found that mono-substituted aryl derivatives are alsoactive.

Moreover, compounds of formula (I) as defined above advantageouslyexhibit no activity toward the adrenergic α2A receptor relative to priorart compounds such as Guanabenz (FIG. 4). This loss in alpha-2adrenergic activity renders the compounds therapeutically useful in thetreatment of the disorders associated with protein misfolding stress andin particular with an accumulation of misfolded proteins, such asCharcot Marie Tooth (CMT), retinal diseases, preferably RetinitisPigmentosa (RP), Alzheimer's disease, Parkinson's disease (PD),Amyotrophic Lateral Sclerosis (ALS), Huntington's disease, tauopathies,prion diseases, diabetes, preferably type 2 diabetes and cancer. Theabsence of alpha-2 adrenergic activity means that compounds of formula(I) can be administered at a dosage suitable to treat the aforementioneddiseases, without any significant effect on blood pressure.

A second aspect of the invention relates to a compound of formula (II),or a pharmaceutically acceptable salt thereof,

wherein:R₁ is alkyl, Cl, F or Br;R₂ is H or F;R₃ is selected from H and alkyl;R₄ is selected from H and C(O)R₆;R₅ is H;or R₄ and R₅ are linked to form a heterocyclic group which is optionallysubstituted with one or more R₁₀ groups;R₆ is selected from R₇, OR₇ and NR₈R₉;R₇, R₈ and R₉ are each independently selected from alkyl, cycloalkyl,aralkyl, cycloalkenyl, heterocyclic, aryl and heteroaryl, each of whichis optionally substituted with one or more R₁₀ groups;each R₁₀ is independently selected from halogen, OH, CN, COO-alkyl,aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl,N(alkyl)₂, CF₃, alkyl and alkoxy;X and Z are each independently CR₁₁, and Y is N;R₁₁ is H or F.

A third aspect of the invention relates to a compound of formula (III),or a pharmaceutically acceptable salt thereof,

wherein:R₁ is alkyl, Cl, F or Br;R₂ is H or F;R₃ is selected from H and alkyl;R₄ is C(O)R₆;R₅ is H;or R₄ and R₅ are linked to form a heterocyclic group which is optionallysubstituted with one or more R₁₀ groups;R₆ is selected from R₇, OR₇ and NR₈R₉;R₇, R₈ and R₉ are each independently selected from alkyl, cycloalkyl,aralkyl, cycloalkenyl, heterocyclic, aryl and heteroaryl, each of whichis optionally substituted with one or more R₁₀ groups;each R₁₀ is independently selected from halogen, OH, CN, COO-alkyl,aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl,N(alkyl)₂, CF₃, alkyl and alkoxy;X and Z are each independently CR₁₁, and Y is selected from CR₁₁ and N;andR₁₁ is H or F.

A fourth aspect of the invention relates to a compound of formula (IV),or a pharmaceutically acceptable salt thereof,

wherein:R₁ is alkyl or Br;R₂ is H;R₃ is selected from H and alkyl;R₄ is selected from H and C(O)R₆;R₅ is H;or R₄ and R₅ are linked to form a heterocyclic group which is optionallysubstituted with one or more R₁₀ groups;R₈ is selected from R₇, OR₇ and NR₈R₉;R₇, R₈ and R₉ are each independently selected from alkyl, cycloalkyl,aralkyl, cycloalkenyl, heterocyclyl and aryl, each of which isoptionally substituted with one or more R₁₀ groups;each R₁₀ is independently selected from halogen, OH, CN, COO-alkyl,aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl,N(alkyl)₂, CF₃, alkyl and alkoxy;X and Z are each CH and Y is CR₁₁;R₁₁ is H or F.

A further aspect of the invention relates to pharmaceutical compositionscomprising a compound of formula (II), (III) or (IV) as described above,admixed with a suitable pharmaceutically acceptable diluent, excipientor carrier.

DETAILED DESCRIPTION

As used herein, the term “alkyl” includes both saturated straight chainand branched alkyl groups which may be substituted (mono- or poly-) orunsubstituted. Preferably, the alkyl group is a C₁₋₂₀ alkyl group, morepreferably a C₁₋₁₅, more preferably still a C₁₋₁₂ alkyl group, morepreferably still, a C₁₋₆ alkyl group, more preferably a C₁₋₃ alkylgroup. Particularly preferred alkyl groups include, for example, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl.Suitable substituents include, for example, one or more R¹⁰ groups.Preferably, the alkyl group is unsubstituted.

As used herein, the term “cycloalkyl” refers to a cyclic alkyl groupwhich may be substituted (mono- or poly-) or unsubstituted. Preferably,the cycloalkyl group is a C₃₋₁₂ cycloalkyl group. Suitable substituentsinclude, for example, one or more R¹⁰ groups.

As used herein, the term “alkenyl” refers to a group containing one ormore carbon-carbon double bonds, which may be branched or unbranched,substituted (mono- or poly-) or unsubstituted. Preferably the alkenylgroup is a C₂₋₂₀ alkenyl group, more preferably a C₂₋₁₅ alkenyl group,more preferably still a C₂₋₁₂ alkenyl group, or preferably a C₂₋₆alkenyl group, more preferably a C₂₋₃ alkenyl group. Suitablesubstituents include, for example, one or more R¹⁰ groups as definedabove. The term “cyclic alkenyl” is to be construed accordingly.

As used herein, the term “aryl” refers to a C₆₋₁₂ aromatic group whichmay be substituted (mono- or poly-) or unsubstituted. Typical examplesinclude phenyl and naphthyl etc. Suitable substituents include, forexample, one or more R¹⁰ groups.

As used herein, the term “heterocycle” (also referred to herein as“heterocyclyl” and “heterocyclic”) refers to a substituted (mono- orpoly-) or unsubstituted saturated, unsaturated or partially unsaturatedcyclic group containing one or more heteroatoms selected from N, O andS, and which optionally further contains one or more CO groups. Suitablesubstituents include, for example, one or more R¹⁰ groups. The term“heterocycle” encompasses both heteroaryl groups and heterocycloalkylgroups as defined below.

As used herein, the term “heteroaryl” refers to a C₂₋₁₂ aromatic,substituted (mono- or poly-) or unsubstituted group, which comprises oneor more heteroatoms. Preferably, the heteroaryl group is a C₄₋₁₂aromatic group comprising one or more heteroatoms selected from N, O andS. Suitable heteroaryl groups include pyrrole, pyrazole, pyrimidine,pyrazine, pyridine, quinoline, thiophene, 1,2,3-triazole,1,2,4-triazole, thiazole, oxazole, iso-thiazole, iso-oxazole, imidazole,furan and the like. Again, suitable substituents include, for example,one or more R¹⁰ groups.

As used herein, the term “heterocycloalkyl” refers to a substituted(mono- or poly-) or unsubstituted cyclic aliphatic group which containsone or more heteroatoms. Preferred heterocycloalkyl groups includepiperidinyl, pyrrolidinyl, piperazinyl, thiomorpholinyl and morpholinyl.More preferably, the heterocycloalkyl group is selected fromN-piperidinyl, N-pyrrolidinyl, N-piperazinyl, N-thiomorpholinyl andN-morpholinyl. Again, suitable substituents include, for example, one ormore R¹⁰ groups.

As used herein, the term “aralkyl” includes, but is not limited to, agroup having both aryl and alkyl functionalities. By way of example, theterm includes groups in which one of the hydrogen atoms of the alkylgroup is replaced by an aryl group, e.g. a phenyl group optionallyhaving one or more substituents such as halo, alkyl, alkoxy, hydroxy,and the like. Typical aralkyl groups include benzyl, phenethyl and thelike.

In one preferred embodiment, R₁ is Cl, Br, Me or F, more preferably, Cl.

In one preferred embodiment, R₂ is H.

In one preferred embodiment, Y is CR₁₁.

In another preferred embodiment, Y is N.

In one preferred embodiment, R₃ and R₄ are both H.

In one preferred embodiment, R₃ is H and R₄ is C(O)R₆.

In one preferred embodiment, R₆ is alkyl or alkoxy, more preferably, Meor OMe.

In one preferred embodiment, R₄ and R₅ are linked to form a heterocyclicgroup which is optionally substituted with one or more R₁₀ groups.

In one preferred embodiment, said compound is of formula (Ia), or apharmaceutically acceptable salt thereof,

wherein R₁, R₂, R₃ and R₁₀ are as defined above.

In one especially preferred embodiment, the compound of formula (I) isselected from the following:

and pharmaceutically acceptable salts thereof.

In one highly preferred embodiment, the compound of formula (I) isselected from Examples 1, 3, 6 and 15 as set out above.

Even more preferably, the compound of formula (I) is selected fromExample 1 and Example 15, more preferably Example 1, i.e. the compound1-[(E)-[(2-chlorophenyl) methylidene]amino]-guanidine.

Compounds

One aspect of the invention relates to compounds of formulae (II), (III)or (IV), or pharmaceutically acceptable salts thereof, as defined above.Preferred aspects of the invention apply mutatis mutandis. Particularlypreferred compounds for this aspect of the invention include Examples 7,8, 9, 13 and 16 as described herein.

Therapeutic Applications

The Applicant has demonstrated that compounds of formula (I) havepotential therapeutic applications in treating disorders associated withaccumulation of misfolded proteins. In particular, compounds of formula(I) have been shown to have a protective effect against cytotoxicendoplasmic reticulum (ER) stress and age related disorders.

Another aspect of the invention relates to the use of a compound offormula (I) as defined above in the preparation of a medicament fortreating a disorder associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins.

As used herein the phrase “preparation of a medicament” includes the useof one or more of the above described compounds directly as themedicament in addition to its use in a screening programme for furtheractive agents or in any stage of the manufacture of such a medicament.

Yet another aspect of the invention relates to a method of treating adisorder associated with protein misfolding stress and in particularwith an accumulation of misfolded proteins in a subject in need thereof,said method comprising administering a therapeutically effective amountof a compound of formula (I) as defined above to said subject.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

Herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease ordisorder, substantially ameliorating clinical symptoms of a disease ordisorder or substantially preventing the appearance of clinical symptomsof a disease or disorder.

The term “therapeutically effective amount” refers to that amount of thecompound being administered which will relieve to some extent one ormore of the symptoms of the disease or disorder being treated.

The unfolded protein response (UPR) is a component of the cellulardefence system against misfolded proteins that adapts folding in theendoplasmic reticulum (ER) to changing conditions. The UPR is activatedin response to an accumulation of unfolded or misfolded proteins in thelumen of the endoplasmic reticulum. In this scenario, the UPR has twoprimary aims: (i) to restore normal function of the cell by haltingprotein translation, and (ii) to activate the signaling pathways thatlead to the increased production of molecular chaperones involved inprotein folding. If these objectives are not achieved within a certaintimeframe, or the disruption is prolonged, the UPR aims towardsapoptosis.

Upstream components of the UPR are the ER-resident trans-membraneproteins IRE1, ATF6, and PERK, which sense folding defects to reprogramtranscription and translation in a concerted manner and restoreproteostasis. Activated IRE1 and ATF6 increase the transcription ofgenes involved in ER folding, such as those encoding the chaperones BiPand GRP94. Activated PERK attenuates global protein synthesis byphosphorylating the subunit of translation initiation factor 2 (elF2α)on Ser51 while promoting translation of the transcription factor ATF4.The latter controls expression of CHOP, another transcription factor,which in turn promotes expression of PPP1R15A/GADD34. PPP1R15A, aneffector of a negative feedback loop that terminates UPR signaling,recruits a catalytic subunit of protein phosphatase 1 (PP1c) todephosphorylate elF2α, allowing protein synthesis to resume. UPR failurecontributes to many pathological conditions that might be corrected byadequate boost of this adaptive response. Selective inhibitors of thestressed-induced elF2α phosphatase PPP1R15A-PP1 delays elF2αdephosphorylation and consequently protein synthesis selectively instressed cells, without affecting protein synthesis in unstressed cells.This prolongs the beneficial effects of the UPR. A transient reductionof protein synthesis is beneficial to stressed cells because decreasingthe flux of proteins synthetized increases the availability ofchaperones and thus protects from misfolding stress (P. Tsaytler, H. P.Harding, D. Ron and A. Bertolotti, Science, 332, 1 Apr. 2011, 91-94).Non-selective inhibitors of the 2 elF2α phosphatases might haveundesirable effects, as persistent translation inhibition isdeleterious. Indeed, genetic ablation of both PPP1R15A and PPP1R15Bresults in early embryonic lethality in mice indicating that inhibitionof the two elF2α phosphatases PPP1R15A-PP1 and PPP1R15B-PP1 isdeleterious in an organismal context. In contrast, genetic ablation ofPPP1R15A has no harmful consequence in mice (Harding et al., 2009, ProcNatl Acad Sci USA, 106, 1832-1837). Furthermore, specific inhibitors ofPPP1R15A are predicted to be inert in unstressed cells, as the PPP1R15Ais not expressed in absence of stress. Thus, selective PPP1R15Ainhibitors are predicted to be safe. Non-selective inhibitors of the twoelF2α phosphatases may also be useful to treat protein misfoldingdiseases, when used at doses that result in only a partial inhibition ofthe phosphatases.

Cytoprotection against ER stress can be measured by a suitable assay.For example, cytoprotection can be measured in HeLa cells in which ERstress is elicited by the addition of media containing tunicamycin, amixture of homologous nucleoside antibiotics that inhibits theUDP-HexNAc: polyprenol-P HexNAc-1-P family of enzymes and is used toinduce unfolded protein response. Cell viability can be detected in thepresence and absence of inhibitor compounds after a set period of time,by measuring the reduction of WST-8 into formazan using a standard cellviability kit (such as Cell Viability Counting Kit-8 from Dojindo).Cytoprotection from ER stress is measured in terms of the percentageincrease in viable cells (relative to control) after ER stress. Furtherdetails of a suitable assay are set forth in the accompanying Examplessection.

In one preferred embodiment, the compound of formula (I) is capable ofprolonging the protective effect of the UPR relative to the control(i.e. in the absence of inhibitor compound) by at least 20%, morepreferably, at least 30%, even more preferably, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, more preferably still, atleast 90%.

The Applicant has demonstrated that compounds of formula (I) areinhibitors of PPP1R15A-PP1 interaction which induce a protective effect.Preferably, the compound exhibits a protective effect with EC₅₀ of lessthan about 5 μM, even more preferably, less than about 2 μM, morepreferably still, less than about 1 μM. The compound should preferablybe devoid of alpha2 adrenergic activity. Thus, in one preferredembodiment the compound does not exhibit any activity in a functionalalpha-2-adrenergic assay.

The Applicant has further demonstrated that certain compounds of formula(I) selectively inhibit PPP1R15A-PP1, and thus prolong the protectiveeffect of the UPR, thereby rescuing cells from protein misfoldingstress. Inhibitors of PPP1R15A-PP1 described in the present inventiontherefore have therapeutic applications in the treatment of a variety ofdiseases associated with protein misfolding stress and in particularwith an accumulation of misfolded proteins.

In one embodiment, the compound of formula (I) is capable of inhibitingPPP1R15A and PPP1R15B.

In one preferred embodiment, the compound of formula (I) is capable ofselectively inhibiting PPP1R15A over PPP1R15B.

In one preferred embodiment of the invention, the compound of formula(I) is for use in treating neurodegenerative diseases, and morespecifically where accumulation of misfolded proteins is involved in themode of action (Brown et al, 2012, Frontiers in Physiology, 3, Article263).

In one particularly preferred embodiment, the compound of formula (I) isfor use in treating a disorder selected from Charcot Marie Tooth, severeDejerine-Sottas syndrome (Voermans et al., 2012, J Peripher New Syst,17(2), 223-5), a retinal disease (such as but not restricted toretinitis pigmentosa, retinal ciliopathies, macular degeneration,diabetic retinopathy), Alzheimer's disease, Parkinson's disease,Amyotrophic Lateral Sclerosis (ALS), Huntington's disease, tauopathies,prion diseases, type 2 diabetes and/or type 1 diabetes and cancer, suchas but not restricted to, multiple myeloma.

In one embodiment, the invention relates to a compound of formula (I) asdefined above for use in treating a disorder associated with the elF2αphosphorylation pathway where accumulation of misfolded proteins isinvolved in the mode of action. Preferably, the disorder is aPPP1R15A-related disease or disorder. Examples of such disorders includeprotein misfolding diseases, such as but not limited to, Charcot MarieTooth, severe Dejerine-Sottas syndrome and Retinitis pigmentosa.

In another embodiment, the invention relates to a compound of formula(I) as defined above for use in treating a disorder caused by,associated with or accompanied by elF2α phosphorylation and/or PPP1R15Aactivity where accumulation of misfolded proteins is involved in themode of action.

In another embodiment, the invention relates to a compound of formula(I) as defined above for use in treating UPR disorder such as, but notlimited to aging (Naidoo et al., 2008, J Neurosci, 28, 6539-48).

As used herein, “PPP1R15A related disease or disorder” refers to adisease or disorder characterized by abnormal PPP1R15A activity whereaccumulation of misfolded proteins is involved in the mode of action.Abnormal activity refers to: (i) PPP1R15A expression in cells whichnormally do not express PPP1R15A; (ii) increased PPP1R15A expression;or, (iii) increased PPP1R15A activity.

In another embodiment, the invention relates to a method of treating amammal having a disease state alleviated by the inhibition of PP1R15A,where accumulation of misfolded proteins is involved in the mode ofaction, wherein the method comprises administering to a mammal atherapeutically effective amount of a compound of formula (I) as definedabove.

In another embodiment, the invention relates to a PPP1R15A inhibitor offormula (I) or a pharmaceutical acceptable salt thereof for the use intreating disorders associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins and/or UPRdisorders, wherein said compound has no or reduced adrenergic alpha 2agonist activity in comparison with Guanabenz.

In another embodiment, the invention relates to a PPP1R15A inhibitor offormula (I) or a pharmaceutical acceptable salt thereof for the use intreating disorders associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins and/or UPRdisorders, wherein said compound does not inhibit protein translation innon-stressed cells expressing PPP1R15B.

In another embodiment, the invention relates to a method of treating adisorder characterized by ER stress response activity with anaccumulation of misfolded proteins, the method comprising administeringto a patient a therapeutically effective amount of at least one compoundof formula (I) wherein said compound modulates ER stress response.

In another embodiment, the invention relates to PPP1R15A inhibitor offormula (I) or a pharmaceutical acceptable salt thereof for the use intreating disorders associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins and/or UPRdisorders, wherein said compound has a selectivity towards PPP1R15A-PP1holophosphatase, having but no or reduced activity towards PPP1R15B-PP1holophosphatase, and wherein the ratio (activity towards PPP1R15A-PP1holophosphatase/activity towards PPP1R15B-PP1) for said compound is atleast equal or superior to the ratio (activity towards PPP1R15A-PP1holophosphatase/activity towards PPP1R15B-PP1) for Guanabenz.

In another embodiment, the Invention relates to a PPP1R15A inhibitor offormula (I) or a pharmaceutical acceptable salt thereof for the use intreating disorders associated with protein misfolding stress and inparticular with an accumulation of misfolded proteins and/or UPRdisorders, wherein:

-   -   said compound has an activity towards PPP1R15A-PP1        holophosphatase but no or reduced activity towards PPP1R15B-PP1        holophosphatase, and;    -   wherein the ratio (activity towards PPP1R15A-PP1        holophosphatase/activity towards PPP1R15B-PP1) for said compound        is at least equal or superior to the ratio (activity towards        PPP1R15A-PP1 holophosphatase/activity towards PPP1R15B-PP1) for        Guanabenz; and    -   wherein said compound has no or reduced adrenergic alpha 2        agonist activity in comparison with Guanabenz.

As used herein, the disease or disorder characterized by ER stressresponse activity, and/or the disease or disorder associated withprotein misfolding stress and in particular with an accumulation ofmisfolded proteins and/or UPR disorders, is selected from Charcot MarieTooth, severe Dejerine-Sottas syndrome (Voermans et al., 2012, JPeripher New Syst, 17(2), 223-5), a retinal disease (such as but notrestricted to retinitis pigmentosa, retinal ciliopathies, maculardegeneration, diabetic retinopathy), Alzheimer's disease, Parkinson'sdisease, Amyotrophic Lateral Sclerosis (ALS), Huntington's disease,diabetes, such as but not restricted to type 2 diabetes and cancer suchas but not restricted to multiple myeloma.

Charcot Marie Tooth

In one preferred embodiment, the compound of formula (I) is for use intreating Charcot Marie Tooth.

Over a 100 mutations in the gene encoding myelin protein zero (P0), asingle-pass transmembrane protein, which is the major protein producedby myelinating Schwann cells causes Charcot-Marie-Tooth neuropathy(D'Antonio et al., 2009, J Neurosci Res, 87, 3241-9). The mutations aredominantly inherited and cause the disease through a gain of toxicfunction (D'Antonio et al., 2009, J Neurosci Res, 87, 3241-9). Deletionof serine 63 from P0 (P0S63del) causes Charcot-Marie-Tooth 1B neuropathyin humans and a similar demyelinating neuropathy in transgenic mice. Themutant protein accumulates in the ER and induces the UPR (D'Antonio etal., 2009, J Neurosci Res, 87, 3241-9). Genetic ablation of CHOP, apro-apoptotic gene in the UPR restores motor function inCharcot-Marie-Tooth mice (Pennuto et al., 2008, Neuron, 57, 393-405).The finding that PPP1R15A inhibition in cells nearly abolishes CHOPexpression in ER-stressed cells indicates that genetic orpharmacological inhibition of PPP1R15A should reduce motor dysfunctionin Charcot-Marie-Tooth mice. Recently, D'Antonio et al. (2013 J. Exp.Med Vol. pp 1-18) demonstrated that P0S63del mice treated withsalubrinal, a small molecule that increases the phosphorylation ofelF2alpha (Boyce et al. 2005 Science Vol. 307 pp 935-939) regainedalmost normal motor capacity in rotarod analysis and was accompanied bya rescue of morphological and electro-physiological abnormalities.Accumulation of the of CMT-related mutant in the ER proteins is notunique to P0S63del; at least five other P0 mutants have been identifiedthat are retained in the ER and elicit an UPR (Pennuto et al., 2008Neuron Vol. 57 pp 393-405; Saporta et al., 2012 Brain Vol. 135 pp2032-2047). In addition, protein misfolding and accumulation ofmisfolded protein in the ER have been implicated in the pathogenesis ofother CMT neuropathies as a result of mutations in PMP22 and Cx32 (Colbyet al., 2000 Neurobiol. Disease Vol. 7 pp 561-573; Kleopa et al., 2002J. Neurosci. Res. Vol. 68 pp 522-534; Yum et al., 2002 Neurobiol. Dis.Vol. 11 pp 43-52). However, Salubrinal is toxic and can not be used totreat human patients D'Antonio et al. (2013 J. Exp. Med Vol. pp 1-18).In contrast, the PPP1R15A inhibitors of formula (I) are predicted to besafe and could be useful for the treatment of CMT-1A and 1B.

Retinal Diseases

Recently published literature has provided evidences that the UPR isinvolved in the development of retinal degeneration: inherited retinaldegeneration such as retinal ciliopathies & retinitis pigmentosa,macular degeneration, retinopathy of premarurity, light-induced retinaldegeneration, retinal detachment, diabetic retinopathy and glaucoma (forreview Gorbatyuk et Gorbatyuk 2013—Retinal degeneration: Focus on theunfolded protein response, Molecular Vision Vol. 19 pp 1985-1998).

In one preferred embodiment, the compound of formula (i) is for use intreating retinal diseases, more preferably, inherited retinaldegeneration such as retinal ciliopathies & retinitis pigmentosa,macular degeneration, retinopathy of premarurity, light-induced retinaldegeneration, retinal detachment, diabetic retinopathy and glaucoma.

Retinal ciliopathies are a group of rare genetic disorders originatingfrom a defect in the primary cilium of photoreceptors thus inducingretinitis pigmentosa. This defect has been reported to induce an ERstress due to protein accumulation in the inner segment of thephotoreceptor which in turn induces the UPR (WO2013/124484). Retinaldegeneration is a very common feature in ciliopathies that can beobserved either in isolated retinitis pigmentosa such as Leber'scongenital amaurosis or X-linked retinitis pigmentosa, or also insyndromic conditions like the Bardet-Biedl Syndrome (BBS) or the Alstromsyndrome (ALMS). The retinal ciliopathy is selected from the groupconsisting of Bardet-Biedl syndrome, Senior-Loken syndrome, Joubertsyndrome, Salidono-Mainzer syndrome, Sensenbrenner syndrome, Jeunesyndrome, Meckel-Gruder syndrome, Alstrom syndrome, MORM syndrome,Leber's congenital amaurosis caused by mutation in a ciliary gene andX-linked retinitis pigmentosa caused by mutation in the RPGR gene.

Retinitis pigmentosa is an inherited, degenerative eye disease thatcauses severe vision impairment and often blindness. It is the mostcommon cause of genetically determined blindness. Sufferers willexperience one or more of the following symptoms: night blindness;tunnel vision (no peripheral vision); peripheral vision (no centralvision); latticework vision; aversion to glare; slow adjustment fromdark to light environments and vice versa; blurring of vision; poorcolor separation; and extreme tiredness.

Emerging evidence supports a role of ER stress in retinal apoptosis andcell death (Jing et al., 2012, Exp Diabetes Res, 2012, 589589). Retinispigmentosa (RP) is the most common form of hereditary retinaldegeneration caused by over 100 mutations in the rhodopsin gene (Dryjaet al., 1991, Proc Natl Acad Sci USA, 88, 9370-4). Rhodopsin is a Gprotein-coupled receptor that transduces light in the rod photoreceptorsand consists of a covalent complex between the transmembrane proteinopsin of 348 amino acids, covalently bound to 11-cis retinal(Palczewski, 2006, Annu Rev Biochem, 76, 743-67). The RP-causingrhodopsin mutations are mostly missense mutations distributed throughoutthe protein (Dryja et al., 1991, Proc Natl Acad Sci USA, 88, 9370-4),similar to the ALS-causing SOD1 mutations (Valentine et al., 2005, AnnuRev Biochem, 74, 563-93). The RP-causing rhodopsin mutants have beenstudied in diverse systems and results from heterologous expression ofthe proteins in mammalian cells, in transgenic mice and drosophila areconsistent (Griciuc et al., 2011, Trends Mol Med, 17, 442-51). The mostprevalent RP-causing rhodopsin are misfolded, do not bind11-cis-retinal, do not reach the cell surface but are retained in the ER(Griciuc et al., 2011, Trends Mol Med, 17, 442-5.1). Misfolding of therhodopsin mutants causes ER stress and rod cell death (Griciuc et al.,2011, Trends Mol Med, 17, 442-51). This strongly suggests that thePPP1R15A inhibitors described in the invention will be useful to treatRP.

Age-related macular degeneration (AMD) is the main cause of legalblindness among those over 65 years of age in the United States. AMD wasreported to account for 54% of all current cases of blindness among theCaucasian population in the United States. The study predicted that as aresult of the rising prevalence of AMD, the number of blind people inthe US could increase by as much as 70% by 2020. Shen et al. (2011Effect of Guanabenz on Rat AMD Models and Rabbit Choroidal Blood—Vol. 5pp 27-31) demonstrated that Guanabenz significantly protected retinalpigment epithelium (RPE) from NalO3-induced degeneration, inhibited thedevelopment of choroidal neovascularization (CNV) in laser-induced ratAMD model and increased choroidal blood flow markedly in vivo.

However, Guanabenz is an alpha2 adrenergic receptor and because of itshypotensive activity, it can not be used to treat retinal or maculardegeneration.

Compounds of the invention which are PPP1R15A inhibitors like Guanabenzbut which advantageously exhibit no activity toward the adrenergicalpha2A receptor will ameliorate retinal or macular.

Alzheimer's Disease, Parkinson's Disease, ALS, Huntington's Disease,Tauopathies and Prion Diseases

In one preferred embodiment, the compound of formula (I) is for use intreating a disease selected from Alzheimer's disease, Parkinson'sdisease, ALS, Huntington's disease, tauopathies and prion diseases.

Because accumulation of misfolded proteins is a hallmark of diversediseases and having shown that compound of formula (I) reducesaccumulation of 4 unrelated misfolded and disease-causing proteins (FIG.4-6), the compound of formula (I) will be useful to also treat otherneurodegenerative diseases caused by accumulation of misfolded proteins.

In addition, as UPR induction is a hallmark of these diseases caused byaccumulation of misfolded protein, the compound of formula (I) will beuseful to treat these diseases. (Scheper & Hoozemans 2009; Kim et al.2008).

Guanabenz reduces the symptoms of prion infected mice (D.Tribouillard-Tanvier et al., 2008 PLoS One 3, e1981). However, Guanabenzis not useful for the treatment of human protein misfolding diseases dueto its hypotensive activity. In contrast, the PPP1R15A inhibitors,devoid of alpha2 adrenergic activity, and described in this inventioncould be useful to treat prion diseases.

Parkinson's Disease (PD)

Salubrinal inhibits the PPP1R15A mediated dephosphorylation of elF2α(Boyce et al. 2005 Science Vol. 307 pp 935-939). Recently, Colta et al.(J. of Neuroscience 2012 Vol. 32 No 10 pp 3306-3320) demonstrated thatSalubrinal significantly attenuates disease manifestations in two animalmodels of alpha-synucleinopathy.

Without to be bound by a theory, it is anticipated that compounds of theinvention which are PPP1R15A inhibitors will ameliorate diseasemanifestations of alpha-syncleinopathies such as Parkinson's disease.

Amyotrophic Lateral Sclerosis (ALS)

Saxena et al. (Nature Neuroscience 2009 Vol. 12 pp 627-636) demonstratedthat Salubrinal extends the life span of a G93A-SOD1 transgenic mousemodel of motor neuron disease. Without to be bound by a theory, it isanticipated that compounds of the invention which are PPP1R15Ainhibitors will ameliorate disease manifestations of ALS with the SOD1mutation G93A. More than 140, mostly missense, mutations in the SOD1gene cause aggregation of the affected protein in familial forms ofamyotrophic lateral sclerosis (ALS). Because diverse SOD1 mutants sharecommon defects (Munch et al. 2010), it is accepted that diverse SOD1mutant cause ALS by a common mechanism. Moreover, the clinicalmanifestations are shared between sporadic and familial forms of thediseases, and it is now well recognized that protein misfolding plays acentral role in both familial and sporadic ALS. Therefore, the compoundsof formula (I) can be used to treat both familial and sporadic forms ofALS.

The Applicant has found that the cytoprotective activity of guanabenz onprotein misfolding stress is surprisingly broad as guanabenz alsoreduces mutant huntingtin accumulation in cells (WO 2008/041133). Thisfinding is unexpected since mutant huntingtin is either cytosolic ornuclear. However, there is evidence that mutant huntingtin metabolismhas previously been connected to the ER stress response (Nishitoh etal., 2002, Genes Dev, 16, 1345-55; Rousseau et al., 2004, Proc Natl AcadSci USA, 101, 9648-53; Duennwald and Lindquist, 2008, Genes Dev, 22,3308-19). The Applicant's findings that guanabenz protects cells fromcytotoxic ER stress and reduces mutant huntingtin accumulation furthersupports the idea that there may be aspects of the ER stress responsethat impact on mutant huntingtin accumulation. Furthermore, dysfunctionof the ER stress response has been involved in a variety of pathologies,including type 2 diabetes and neurodegeneration (Scheperand Hoozemans,2009, Curr Med Chem, 16, 615-26). Thus, without wishing to be bound bytheory, it is believed that guanabenz and related compounds have aprotective effect against secondary UPR disorders, namely disorders dueto ah accumulation of a non-ER resident misfolded protein, which inducesthe UPR.

Diabetes

In one preferred embodiment, the compound of formula (I) is for use intreating diabetes, more preferably type 2 diabetes.

The insulin-secreting β-cells in the pancreas have a heavy and tightlyregulated biosynthetic burden consisting in insulin secretion. Thus,these cells have an important need to maintain ER homeostasis (Back andKaufman, 2012, Annu Rev Biochem, 81, 767-93). Type 2 diabetes ismanifested by increased levels of blood glucose due to insulinresistance in the adipose, muscle and liver and/or Impaired insulinsecretion from pancreatic β-cells. As a response, β-cells mass increaseand their function is enhanced. Eventually, the burden on the β-cells istoo high leading to their progressive decline and death. Increasingevidence reveals that death of β-cells results from ER stress (Back andKaufman, 2012, Annu Rev Biochem, 81, 767-93). Importantly, Chop deletionimproves β-cells function in diverse models of diabetes (Song et al.,2008, J Clin Invest, 118, 3378-89). Without wishing to be bound bytheory, it is believed that inhibitors of PPP1R15A-PP1 will improveβ-cells function in type 2 diabetes since inhibition of PPP1R15A-PP1reduces the levels of the pro-apoptotic protein CHOP during ER stress(Tsaytler et al., 2011, Science, 332, 91-4).

Cancer

In one preferred embodiment, the compound of formula (I) is for use intreating cancer.

Cancer cells have high metabolic requirement and their proliferationrelies on efficient protein synthesis. Translation initiation plays acrucial role in controlling protein homeostasis, differentiation,proliferation and malignant transformation. Increasing translationinitiation contributes to cancer initiation and conversely, decreasingtranslation initiation could reduce tumor growth (Donze et al., 1995,EMBO J, 14, 3828-34; Pervin et al., 2008, Cancer Res, 68, 4862-74; Chenet al., 2011, Nat Chem Biol, 7, 610-6). Without wishing to be bound bytheory, it is believed that inhibiting PPP1R15A could selectively reducetranslation in tumor cells and thus reduce tumor growth.

Aging

Aging is known to impair stress responses and in particular, the UPR isimpaired with age (Naidoo et al., 2008, J Neurosci, 28, 6539-48). Thus,prolonging the beneficial effect of the UPR by inhibition of elF2αphosphatase could ameliorate age-related disorders.

Pharmaceutical Compositions

For use according to the present invention, the compounds orphysiologically acceptable salts, esters or other physiologicallyfunctional derivatives thereof, described herein, may be presented as apharmaceutical formulation, comprising the compounds or physiologicallyacceptable salt, ester or other physiologically functional derivativethereof, together with one or more pharmaceutically acceptable carrierstherefore and optionally other therapeutic and/or prophylacticingredients. The carriers) must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. The pharmaceutical compositionsmay be for human or animal usage in human and veterinary medicine.

Examples of such suitable excipients for the various different forms ofpharmaceutical compositions described herein may be found in the“Handbook of Pharmaceutical Excipients, 2^(nd) Edition, (1994), Editedby A Wade and P J Weller.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examplesof suitable diluents include ethanol, glycerol and water.

The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as, or In addition to, the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s), buffer(s), flavouring agent(s), surface activeagent(s), thickener(s), preservative(s) (including anti-oxidants) andthe like, and substances included for the purpose of rendering theformulation isotonic with the blood of the intended recipient.

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like.

Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesInclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal, ocular and sublingual), rectal or parenteral(including subcutaneous, intradermal, intramuscular and intravenous),nasal, intra-ocularly and pulmonary administration e.g., by inhalation.The formulation may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All methods include the step of bringinginto association an active compound with liquid carriers or finelydivided solid carriers or both and then, if necessary, shaping theproduct into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein thecarrier is a solid are most preferably presented as unit doseformulations such as boluses, capsules or tablets each containing apredetermined amount of active compound. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine an active compound in a free-flowing form such as apowder or granules optionally mixed with a binder, lubricant, inertdiluent, lubricating agent, surface-active agent or dispersing agent.Moulded tablets may be made by moulding an active compound with an inertliquid diluent. Tablets may be optionally coated and, if uncoated, mayoptionally be scored. Capsules may be prepared by filling an activecompound, either alone or in admixture with one or more accessoryingredients, into the capsule shells and then sealing them in the usualmanner. Cachets are analogous to capsules wherein an active compoundtogether with any accessory ingredient(s) is sealed in a rice paperenvelope. An active compound may also be formulated as dispersiblegranules, which may for example be suspended in water beforeadministration, or sprinkled on food. The granules may be packaged,e.g., in a sachet. Formulations suitable for oral administration whereinthe carrier is a liquid may be presented as a solution or a suspensionin an aqueous or non-aqueous liquid, or as an oil-in-water liquidemulsion.

Formulations for oral administration include controlled release dosageforms, e.g., tablets wherein an active compound is formulated in anappropriate release—controlling matrix, or is coated with a suitablerelease—controlling film. Such formulations may be particularlyconvenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art. The suppositories may beconveniently formed by admixture of an active compound with the softenedor melted carriers) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of an active compound inaqueous or oleaginous vehicles.

Pharmaceutical formulations of the invention are suitable for ophthalmicadministration, in particular for intra-ocular, topical ocular orperi-ocular administration, more preferably for topical ocular orperi-ocular administration.

Injectible preparations may be adapted for bolus injection or continuousinfusion. Such preparations are conveniently presented in unit dose ormulti-dose containers which are sealed after introduction of theformulation until required for use. Alternatively, an active compoundmay be in powder form which is constituted with a suitable vehicle, suchas sterile, pyrogen-free water, before use.

An active compound may also be formulated as long-acting depotpreparations, which may be administered by intramuscular injection or byimplantation, e.g., subcutaneously or intramuscularly. Depotpreparations may include, for example, suitable polymeric or hydrophobicmaterials, or ion-exchange resins. Such long-acting formulations areparticularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavityare presented such that particles containing an active compound anddesirably having a diameter in the range of 0.5 to 7 microns aredelivered in the bronchial tree of the recipient. As one possibilitysuch formulations are in the form of finely comminuted powders which mayconveniently be presented either in a pierceable capsule, suitably of,for example, gelatin, for use in an inhalation device, or alternativelyas a self-propelling formulation comprising an active compound, asuitable liquid or gaseous propellant and optionally other ingredientssuch as a surfactant and/or a solid diluent. Suitable liquid propellantsinclude propane and the chlorofluorocarbons, and suitable gaseouspropellants include carbon dioxide. Self-propelling formulations mayalso be employed wherein an active compound is dispensed in the form ofdroplets of solution or suspension.

Such self-propelling formulations are analogous to those known in theart and may be prepared by established procedures. Suitably they arepresented in a container provided with either a manually-operable orautomatically functioning valve having the desired spraycharacteristics; advantageously the valve is of a metered typedelivering a fixed volume, for example, 25 to 100 microlitres, upon eachoperation thereof.

As a further possibility an active compound may be in the form of asolution or suspension for use in an atomizer or nebuliser whereby anaccelerated airstream or ultrasonic agitation is employed to produce afine droplet mist for inhalation.

Formulations suitable for nasal administration include preparationsgenerally similar to those described above for pulmonary administration.When dispensed such formulations should desirably have a particlediameter in the range 10 to 200 microns to enable retention in the nasalcavity; this may be achieved by, as appropriate, use of a powder of asuitable particle size or choice of an appropriate valve. Other suitableformulations include coarse powders having a particle diameter in therange 20 to 500 microns, for administration by rapid inhalation throughthe nasal passage from a container held close up to the nose, and nasaldrops comprising 0.2 to 5% w/v of an active compound in aqueous or oilysolution or suspension.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Additionally, such pharmaceuticallyacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Preservatives and other additives mayalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, inert gases and the like.

Formulations suitable for topical formulation may be provided forexample as gels, creams or ointments. Such preparations may be appliede.g. to a wound or ulcer either directly spread upon the surface of thewound or ulcer or carried on a suitable support such as a bandage,gauze, mesh or the like which may be applied to and over the area to betreated.

Liquid or powder formulations may also be provided which can be sprayedor sprinkled directly onto the site to be treated, e.g. a wound orulcer. Alternatively, a carrier such as a bandage, gauze, mesh or thelike can be sprayed or sprinkle with the formulation and then applied tothe site to be treated.

According to a further aspect of the invention, there is provided aprocess for the preparation of a pharmaceutical or veterinarycomposition as described above, the process comprising bringing theactive compound(s) into association with the carrier, for example byadmixture.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active agent with liquid carriers orfinely divided solid carriers or both, and then if necessary shaping theproduct. The invention extends to methods for preparing a pharmaceuticalcomposition comprising bringing a compound of general formula (I) inconjunction or association with a pharmaceutically or veterinarilyacceptable carrier or vehicle.

Salts/Esters

The compounds of the invention can be present as salts or esters, inparticular pharmaceutically and veterinarily acceptable salts or esters.

Pharmaceutically acceptable salts of the compounds of the inventioninclude suitable acid addition or base salts thereof. A review ofsuitable pharmaceutical salts may be found in Berge et al, J Pharm Sci,66, 1-19 (1977). Salts are formed, for example with strong inorganicacids such as mineral acids, e.g. hydrohalic acids such ashydrochloride, hydrobromide and hydroiodide, sulfuric acid, phosphoricacid sulphate, bisulphate, hemisulphate, thiocyanate, persulphate andsulphonic acids; with strong organic carboxylic acids, such asalkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted orsubstituted (e.g., by halogen), such as acetic acid; with saturated orunsaturated dicarboxylic acids, for example oxalic, malonic, succinic,maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylicacids, for example ascorbic, glycolic, lactic, malic, tartaric or citricacid; with amino acids, for example aspartic or glutamic acid; withbenzoic acid; or with organic sulfonic acids, such as (C₁-C₄)-alkyl- oraryl-sulfonic acids which are unsubstituted or substituted (for example,by a halogen) such as methane- or p-toluene sulfonic acid. Salts whichare not pharmaceutically or veterinarily acceptable may still bevaluable as intermediates.

Preferred salts include, for example, acetate, trifluoroacetate,lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate,adipate, alginate, aspartate, benzoate, butyrate, digluconate,cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate,hexanoate, fumarate, nicotinate, palmoate, pectinate,3-phenylpropionate, picrate, pivalate, proprionate, tartrate,lactobionate, pivolate, camphorate, undecanoate and succinate, organicsulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids.

Esters are formed either using organic acids or alcohols/hydroxides,depending on the functional group being esterified. Organic acidsinclude carboxylic acids, such as alkanecarboxylic acids of 1 to 12carbon atoms which are unsubstituted or substituted (e.g., by halogen),such as acetic acid; with saturated or unsaturated dicarboxylic acid,for example oxalic, malonic, succinic, maleic, fumaric, phthalic ortetraphthalic; with hydroxycarboxylic acids, for example ascorbic,glycolic, lactic, malic, tartaric or citric acid; with amino acids, forexample aspartic or glutamic acid; with benzoic acid; or with organicsulfonic acids, such as (C₁-C₄)-alkyl- or aryl-sulfonic acids which areunsubstituted or substituted (for example, by a halogen) such asmethane- or p-toluene sulfonic acid. Suitable hydroxides includeinorganic hydroxides, such as sodium hydroxide, potassium hydroxide,calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcoholsof 1-12 carbon atoms which may be unsubstituted or substituted, e.g. bya halogen).

Enantiomers/Tautomers

In all aspects of the present invention previously discussed, theinvention includes, where appropriate all enantiomers, diastereoisomersand tautomers of the compounds of the invention. The person skilled inthe art will recognise compounds that possess optical properties (one ormore chiral carbon atoms) or tautomeric characteristics. Thecorresponding enantiomers and/or tautomers may be isolated/prepared bymethods known in the art. Enantiomers are characterised by the absoluteconfiguration of their chiral centres and described by the R- andS-sequencing rules of Cahn, Ingold and Prelog. Such conventions are wellknown in the art (e.g. see ‘Advanced Organic Chemistry’, 3^(rd) edition,ed. March, J., John Wiley and Sons, New York, 1985).

Compounds of formula (I) thus also include the tautomer forms offormula:

As an illustrative example, a tautomer form of example 1 is:

Compounds of the invention containing a chiral centre may be used as aracemic mixture, an enantiomerically enriched mixture, or the racemicmixture may be separated using well-known techniques and an individualenantiomer may be used alone.

Stereo and Geometric Isomers

Some of the compounds of the invention may exist as stereoisomers and/orgeometric isomers—e.g. they may possess one or more asymmetric and/orgeometric centres and so may exist in two or more stereoisomeric and/orgeometric forms. The present invention contemplates the use of all theindividual stereoisomers and geometric isomers of those inhibitoragents, and mixtures thereof. The terms used in the claims encompassthese forms, provided said forms retain the appropriate functionalactivity (though not necessarily to the same degree).

The present invention also includes all suitable isotopic variations ofthe agent or a pharmaceutically acceptable salt thereof. An isotopicvariation of an agent of the present invention or a pharmaceuticallyacceptable salt thereof is defined as one in which at least one atom Isreplaced by an atom having the same atomic number but an atomic massdifferent from the atomic mass usually found in nature. Examples ofisotopes that can be incorporated into the agent and pharmaceuticallyacceptable salts thereof include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorus, sulfur, fluorine and chlorine such as ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl, respectively. Certainisotopic variations of the agent and pharmaceutically acceptable saltsthereof, for example, those in which a radioactive isotope such as ³H or¹⁴C is incorporated, are useful in drug and/or substrate tissuedistribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with isotopes such as deuterium,i.e., ²H, may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example, increased in vivo half-life orreduced dosage requirements and hence may be preferred in somecircumstances. For example, the invention includes compounds of generalformula (I) where any hydrogen atom has been replaced by a deuteriumatom. Isotopic variations of the agent of the present invention andpharmaceutically acceptable salts thereof of this invention cangenerally be prepared by conventional procedures using appropriateisotopic variations of suitable reagents.

Prodrugs

The invention further includes the compounds of the present invention inprodrug form, i.e. covalently bonded compounds which release the activeparent drug according to general formula (I) in vivo. Such prodrugs aregenerally compounds of the invention wherein one or more appropriategroups have been modified such that the modification may be reversedupon administration to a human or mammalian subject. Reversion isusually performed by an enzyme naturally present in such subject, thoughit is possible for a second agent to be administered together with sucha prodrug in order to perform the reversion in vivo. Examples of suchmodifications include ester (for example, any of those described above),wherein the reversion may be carried out be an esterase etc. Other suchsystems will be well known to those skilled in the art.

Solvates

The present invention also includes solvate forms of the compounds ofthe present invention. The terms used in the claims encompass theseforms.

Polymorphs

The invention further relates to the compounds of the present inventionin their various crystalline forms, polymorphic forms and (an)hydrousforms. It is well established within the pharmaceutical industry thatchemical compounds may be isolated in any of such forms by slightlyvarying the method of purification and or isolation form the solventsused in the synthetic preparation of such compounds.

Administration

The pharmaceutical compositions of the present invention may be adaptedfor rectal, nasal, intrabronchial, topical (including buccal, sublingualand ophthalmic administration, in particular for intra-ocular, topicalocular or peri-ocular administration), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intraarterial andintradermal), intraperitoneal or intrathecal administration. Preferablythe formulation is an orally administered formulation. The formulationsmay conveniently be presented in unit dosage form, i.e., In the form ofdiscrete portions containing a unit dose, or a multiple or sub-unit of aunit dose. By way of example, the formulations may be in the form oftablets and sustained release capsules, and may be prepared by anymethod well known in the art of pharmacy.

Formulations for oral administration in the present invention may bepresented as: discrete units such as capsules, gellules, drops, cachets,pills or tablets each containing a predetermined amount of the activeagent; as a powder or granules; as a solution, emulsion or a suspensionof the active agent in an aqueous liquid or a non-aqueous liquid; or asan oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or asa bolus etc. Preferably, these compositions contain from 1 to 250 mg andmore preferably from 10-100 mg, of active ingredient per dose.

For compositions for oral administration (e.g. tablets and capsules),the term “acceptable carrier” includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid; talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring and the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally be coated or scored and may be formulatedso as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally, intra-ocularly,topical, peri-ocularly or intramuscularly, and which are prepared fromsterile or sterilisable solutions. Injectable forms typically containbetween 10-1000 mg, preferably between 10-250 mg, of active ingredientper dose.

The pharmaceutical compositions of the present invention may also be inform of suppositories, pessaries, suspensions, emulsions, lotions,ointments, creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredient can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredient can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific compound employed, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and theindividual undergoing therapy. The dosages disclosed herein areexemplary of the average case. There can of course be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

In accordance with this invention, an effective amount of a compound ofgeneral formula (I) may be administered to target a particular conditionor disease. Of course, this dosage amount will further be modifiedaccording to the type of administration of the compound. For example, toachieve an “effective-amount” for acute therapy, parenteraladministration of a compound of general formula (I) is preferred. Anintravenous infusion of the compound in 5% dextrose in water or normalsaline, or a similar formulation with suitable excipients, is mosteffective, although an intramuscular bolus injection is also useful.Typically, the parenteral dose will be about 0.01 to about 100 mg/kg;preferably between 0.1 and 20 mg/kg, in a manner to maintain theconcentration of drug in the plasma at an effective concentration Thecompounds may be administered one to four times daily at a level toachieve a total daily dose of about 0.4 to about 400 mg/kg/day. Theprecise amount of an inventive compound which is therapeuticallyeffective, and the route by which such compound is best administered, isreadily determined by one of ordinary skill in the art by comparing theblood level of the agent to the concentration required to have atherapeutic effect.

The compounds of this invention may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto achieve one or more of the therapeutic indications disclosed herein.Typically, a pharmaceutical composition containing the compound isadministered at an oral dose of between about 0.1 to about 50 mg/kg in amanner consistent with the condition of the patient. Preferably the oraldose would be about 0.1 to about 20 mg/kg.

No unacceptable toxicological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention. The compounds of this invention, which may have goodbioavailability, may be tested in one of several biological assays todetermine the concentration of a compound which is required to have agiven pharmacological effect.

Combinations

In a particularly preferred embodiment, the one or more compounds of theinvention are administered in combination with one or more other activeagents, for example, existing drugs available on the market. In suchcases, the compounds of the invention may be administered consecutively,simultaneously or sequentially with the one or more other active agents.

Drugs in general are more effective when used in combination. Inparticular, combination therapy is desirable in order to avoid anoverlap of major toxicities, mechanism of action and resistancemechanism(s). Furthermore, it is also desirable to administer most drugsat their maximum tolerated doses with minimum time intervals betweensuch doses. The major advantages of combining drugs are that it maypromote additive or possible synergistic effects through biochemicalinteractions and also may decrease the emergence of resistance.

Beneficial combinations may be suggested by studying the inhibitoryactivity of the test compounds with agents known or suspected of beingvaluable in the treatment of a particular disorder. This procedure canalso be used to determine the order of administration of the agents,i.e. before, simultaneously, or after delivery. Such scheduling may be afeature of all the active agents identified herein.

Assay

A further aspect of the invention relates to the use of a compound asdescribed above in an assay for identifying further candidate compoundscapable of inhibiting PPP1R15A-PP1.

Preferably, the assay is a competitive binding assay.

More preferably, the competitive binding assay comprises contacting acompound of the invention with PPP1R15A-PP1 and a candidate compound anddetecting any change in the interaction between the compound accordingto the invention and the PPP1R15A-PP1.

Preferably, the candidate compound is generated by conventional SARmodification of a compound of the invention.

As used herein, the term “conventional SAR modification” refers tostandard methods known in the art for varying a given compound by way ofchemical derivatisation.

Thus, in one aspect, the identified compound may act as a model (forexample, a template) for the development of other compounds. Thecompounds employed in such a test may be free in solution, affixed to asolid support, borne on a cell surface, or located intracellularly. Theabolition of activity or the formation of binding, complexes between thecompound and the agent being tested may be measured.

The assay of the present invention may be a screen, whereby a number ofagents are tested. In one aspect, the assay method of the presentinvention is a high through-put screen.

This invention also contemplates the use of competitive drug screeningassays in which neutralising antibodies capable of binding a compoundspecifically compete with a test compound for binding to a compound.

Another technique for screening provides for high throughput screening(HTS) of agents having suitable binding affinity to the substances andis based upon the method described in detail in WO 84/03564.

It is expected that the assay methods of the present invention will besuitable for both small and large-scale screening of test compounds aswell as in quantitative assays.

Preferably, the competitive binding assay comprises contacting acompound of the invention with PPP1R15A-PP1 in the presence of a knownsubstrate of PPP1R15A-PP1 and detecting any change in the interactionbetween said PPP1R15A-PP1 and said known substrate.

A further aspect of the invention provides a method of detecting thebinding of a ligand to PPP1R15A-PP1, said method comprising the stepsof:

(i) contacting a ligand with PPP1R15A-PP1 in the presence of a knownsubstrate

(ii) detecting any change in the interaction between PPP1R15A-PP1 andsaid known substrate;

and wherein said ligand is a compound of the invention.

One aspect of the invention relates to a process comprising the stepsof:

(a) performing an assay method: described hereinabove;

(b) identifying one or more ligands capable of binding to a ligandbinding domain;

and

(c) preparing a quantity of said one or more ligands.

Another aspect of the invention provides a process comprising the stepsof:

(a) performing an assay method described hereinabove;

(b) identifying one or more ligands capable of binding to a ligandbinding domain; and

(c) preparing a pharmaceutical composition comprising said one or moreligands.

Another aspect of the invention provides a process comprising the stepsof:

(a) performing an assay method described hereinabove;

(b) identifying one or more ligands capable of binding to a ligandbinding domain;

(c) modifying said one or more ligands capable of binding to a ligandbinding domain;

(d) performing the assay method described hereinabove;

(e) optionally preparing a pharmaceutical composition comprising saidone or more ligands.

The invention also relates to a ligand identified by the methoddescribed hereinabove.

Yet another aspect of the invention relates to a pharmaceuticalcomposition comprising a ligand identified by the method describedhereinabove.

Another aspect of the invention relates to the use of a ligandidentified by the method described hereinabove in the preparation of apharmaceutical composition for use in the treatment of a disorderassociated with accumulation of misfolded proteins as defined above.

The above methods may be used to screen for a ligand useful as aninhibitor of PPP1R15A-PP1.

Compounds of general formula (I) are useful both as laboratory tools andas therapeutic agents. In the laboratory certain compounds of theinvention are useful in establishing whether a known or newly discoveredtarget contributes a critical or at least significant biochemicalfunction during the establishment or progression of a disease state, aprocess commonly referred to as ‘target validation’.

The present invention is further described with reference to thefollowing figures, wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dose dependent protection of HeLa cells by Compound of theformula (I), Example 1 of the invention, from ER stress induced by 6hour exposure to tunicamycin. See description test 1.

FIG. 2 shows that Compound of the formula (I), Example 1 of theinvention, postpones translation recovery in stressed cells. Morespecifically, FIG. 2 shows that translation is attenuated 2 h followingTunicamycin addition. Translation recovery is noticeable in ceilstreated with tunicamycin only. Example 1 of the invention prolongstranslation attenuation in tunicamycin treated cells. See descriptiontest 3.

FIG. 3 shows Compound of the formula (I), Example 1 of the invention,unlike Guanabenz, has a no activity for adrenergic α2A receptor asmeasured by a functional assay for the adrenergic α2A receptor. Seedescription test 5.

FIG. 4 shows that a Compound of the formula (I), Example 1 of theinvention prevents ER-retention of P0S63del, the mutant proteinassociated with Charcot Marie Tooth 1B. Y axis: number of cells. UT:untreated.

FIG. 5 shows that a Compound of the formula (I), Example 1 of theinvention reduces accumulation of two unrelated disease-causing,misfolded proteins: mutant huntingtin amino-terminal fragment (Htt48Q)associated with Huntington's disease and SOD1 mutant (A4V), associatedwith amyotrophic lateral sclerosis. Y axis: percentage accumulation ofprotein, relative to untreated cells. UT: untreated.

FIG. 6 shows that a Compound of the formula (I), Example 1 of theinvention reduces accumulation of rhodopsin mutant P23H associated withretinitis pigmentosa. Y axis: number of cells. UT: untreated.

The present invention is further described with reference to thefollowing non-limiting examples.

EXAMPLES

Methods & Materials

Example 1 was purchased from Chemdiv ref: 1683-6588

Example 2 was purchased from Chembridge ref: 5173161

Example 4 was purchased from Enamine ref: Z49562642

Example 6 was purchased from Chemdiv ref: 1683-6502

Preparation of the Compounds According to the Present Invention

The reactants and commercials compounds were purchased from AcrosOrganics, Sigma-Aldrich. The compounds according to the presentinvention can be prepared according to the following general procedure:

General Procedure A:

To a solution of benzaldehyde (1 eq.) in ethanol (300 ml) wassequentially added Aminoguanidine hydrochloride (1 eq.) and sodiumacetate (1 eq.) at 25° C. The resulting reaction mixture was heated at80° C. for next ˜6 hours. Reaction completion was monitored on TLC usingdichloromethane/methanol (8/2) as mobile phase. After completion ofreaction, the reaction mixture was allowed to cool down to 25° C. anddumped in the saturated solution of NaHCO₃ (700 ml). The resultingprecipitate were filtered off under vacuum and washed with water (100ml). The resulting solid material was titurated with diethytether (2×25ml) and dried under vacuum to provide the desired substitutedaminoguanidine derivative.

The following compounds were prepared according general procedure A:

Example 1: 1-[(E)-[(2-chlorophenyl)methylidene]amino]-guanidine

Prepared following general procedure A from 2-chlorobenzaldehyde. ¹H-NMR(DMSO-d₆): δ (ppm) 5.61 (s, 2H); 6.06 (s, 2H); 7.22-7.32 (m, 2H); 7.40(dd, 1H); 8.15 (dd, 1H); 8.28 (s, 1H); MS (ESI+): m/z=197.4 [M+H]⁺

Example 3: 1-[(E)-[(2-fluorophenyl)methylidene]amino]-guanidine

Prepared following general procedure A from 2-fluorobenzaldehyde.

Example 7: 1-[(E)-[(2-chloro-4-fluorophenyl)methylidene]amino]guanidine

Prepared following general procedure A from2-chloro-4-fluorobenzaldehyde in 67% yield. ¹H-NMR (DMSO-d₆): δ (ppm)5.80 (brs, 2H); 5.84 (brs, 2H); 7.19-7.34 (m, 4H); 8.16 (s, 1H); MS(ESI+): m/z=215.1 [M+H]⁺

Example 13: 1-[(E)-[(3-chloropyridin-4-yl)methylidene]amino]guanidine

Prepared following general procedure A from 3-chloroisonicotinaldehydein 50% yield. ¹H-NMR (DMSO-d₆): δ (ppm) 6.01 (brs, 2H); 6.33 (brs, 2H);8.10 (d, 1H); 8.14 (s, 1H); 8.37 (dd, 1H); 8.52 (s, 1H); MS (ESI+):m/z=198.4 [M+H]⁺

Example 15: 1-[(E)-[(2-chloro-6-fluorophenyl)methylidene]amino]guanidine

Prepared following general procedure A from 2-chloronicotinaldehyde in56% yield. ¹H-NMR (DMSO-d₆): δ (ppm) 5.84 (brs, 2H); 5.88 (brs, 2H);7.18-7.35 (m, 3H); 8.16 (s, 1H); MS (ESI+): m/z=215.4 [M+H]⁺.

Intermediate 1:3-chloro-5-fluoroisonicotinaldehyde

To a stirred solution of N,N-Diisopropylamine (0.864 g, 0.006690 mol) inTHF (6 ml) was added n-buLi (1.6M in hexane) (7.6 ml, 0.012164 mol)dropwise over a period of 15 minutes at −78° C. The resulting reactionmixture was stirred at −78° C. for 15 minutes and then it was allowed towarm at 0° C. whereby it was further stirred for 1 hour. The resultingreaction mixture was again cooled at −78° C. and a solution of3-chloro-5-fluoropyridine (0.8 g, 0.006082 mol) in THF (6 ml) was addeddropwise over period of 10 minutes. The resulting reaction mixture wasstirred at −78° C. for 1 hour, thereafter methyl formate (0.73 g,0.012164 mol) was added dropwise at −78° C. The resulting reactionmixture was further stirred at −78° C. for 1 more hour. The reaction wasmonitored on TLC using Hexane:ethylaceate (5:5) as mobile phase. Aftercompletion of reaction, the reaction mixture was dumped in saturatedsolution of NH₄Cl (50 ml) and extracted with Ethyl acetate (4×25 ml).The combined organic extract was washed with demineralised water (50ml), brine (25 ml), dried over sodium sulphate and concentrated undervacuo. Distillation of the organic layer provided the desired aldehyde(0.6 g, 61.85% yield) in crude form. This crude compound was directlyused for the next step without any further treatment.

Example 16:1-[(E)-[(3-chloro-5-fluoropyridin-4-yl)methylidene]amino]guanidine

Prepared following general procedure A from3-chloro-5-fluoroisonicotinaldehyde in 14% yield. ¹H-NMR (DMSO-d₆): δ(ppm) 5.95-6.30 (m, 4H); 8.10 (s, 1H); 8.46-8.52 (m, 2H); MS (ESI+):m/z=216.0 [M+H]⁺.

Example 8:N-{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}acetamide

To a solution of 1-[(E-[(2-chlorophenyl)methylidene]amino]-guanidine(0.50 g, 0.002543 mol) in DMSO (10 ml) was added acetic anhydride (0.26g, 0.002543 mol) at 25° C. The resulting reaction mixture was stirred at25° C. for next 15 hours. Reaction completion was monitored on TLC usingdichloromethane/Methanol (9.5/0.5) as mobile phase. After completion ofreaction the reaction mixture was dumped in the water (100 ml) andextracted with ethyl acetate (2×150 ml). The combined organic extractwas washed with brine (100 ml), dried over sodium sulphate, filtered andconcentrated in vacuo. The resulting crude material was further purifiedby flash chromatography using dichloromethane: methanol as mobile phasewhereby the desired product eluted at around 1.0% methanol indichloromethane. Distillation of the pure product fractions providedN-{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}acetamide(0.080 g, 13% yield). ¹H-NMR (DMSO-d₆): δ (ppm) 2.97 (s, 3H); 7.25-7.41(m, 3H); 7.42-7.53 (m, 1H); 7.79 (brs, 1H); 8.22-8.29 (m, 1H); 8.48 (s,1H); 10.58 (brs, 1H); MS (ESI+): m/z=239.2 [M+H]⁺.

Example 9: methylN-{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}carbamate

To a suspension of 1-[(E)-[(2-chlorophenyl)methylidene]amino]-guanidine(0.15 g, 0.000762 mol) in dichloromethane (5 ml) was added triethylamine(0.32 ml, 0.002288 mol) at 25° C. The resulting reaction mixture wascooled to 0° C. using ice/salt bath; thereafter methylchloroformate(0.09 ml, 0.001144 mol) was added in to the reaction mixture at 0° C.the resulting reaction mixture was stirred at room temperature for 15hours. Reaction completion was monitored on TLC usingdichloromethane/methanol (9/1) as mobile phase. After completion ofreaction, the reaction mixture was dumped in saturated solution ofNaHCO₃ (20 ml) and extracted with dichloromethane (3×25 ml). Thecombined organic extract was washed with D.M. water (20 ml), brine (20ml), dried over sodium sulphate, filtered and concentrated in vacuo. Theresulting crude material was further purified by flash columnchromatography using dichloromethane:methanol as mobile phase wherebythe desired product eluted at around 1.0% methanol in dichloromethane.Distillation of the pure product fractions provided methylN-{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}carbamate(0.065 g, 37% yield). ¹H-NMR (DMSO-d₆): δ (ppm) 3.60 (s, 3H); 7.34-7.43(m, 2H); 7.45-7.52 (m, 1H); 7.67 (brs, 1H); 7.92 (brs, 1H); 8.22-8.30(m, 1H); 8.44 (s, 1H); 11.02 (brs, 1H); MS (ESI+): m/z=255.4 [M+H]⁺.

Selected compounds according to the invention are set forth in Table 1below:

Compound Number Structure Chemical Name Example 1

1-[(E)-[(2- chlorophenyl)methylidene]amino]- guanidine Example 2

1-[(E)-[(2- bromophenyl)methylidene]amino]- guanidine Example 3

1-[(E)-[(2- fluorophenyl)methylidene]amino]- guanidine Example 4

1-[(E)-[(2- methylphenyl)methylidene]amino] guanidine Example 6

2-chlorobenzaldehyde (6-methyl-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)hydrazone Example 7

1-[(E)-[(2-chloro-4- fluorophenyl)methylidene]amino]guanidine Example 8

N-{N-[(E)-[(2- chlorophenyl)methylidene]amino] carbamimidoyl}acetamideExample 9

methyl N-{N-[(E)-[(2- chlorophenyl)methylidene]amino]carbamimidoyl}carbamate Example 13

1-[(E)-[(3-chloropyridin-4- yl)methylidene]amino]guanidine Example 15

1-[(E)-[(2-chloro-6- fluorophenyl)methylidene]amino]guanidine Example 16

1-[(E)-[(3-chloro-5-fluoropyridin-4- yl)methylidene]amino]guanidine

In some of the experiments below, the salt of these compounds may beused; for example, the acetate salt of example 1 formed with acetic acidmay be used.

Cytoprotection from ER Stress (Test 1)

HeLa cells were cultured in Dulbecco's Modified Eagle's Media (DMEM)supplemented with penicillin, streptomycin, containing 5% fetal bovineserum (FBS), at 37° C. in 5% CO₂ atmosphere. Cells were plated in 24well plates at a density of 15,000 cells/ml 24 hours prior treatment. ERstress was elicited by addition of fresh media containing 2.5 μg/mltunicamycin (Sigma-Aldrich) together with eIF2α phosphatases inhibitors(0.2-5 μM). Media were changed 6 h later with fresh media containingphosphatase inhibitors (0.2-5 μM). Inhibitors were dissolved in DMSO (50mM) and DMSO was used as a mock treatment. Cell viability was assessedby measuring the reduction of WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium]into formazan using Cell viability Counting Kit-8 (Dojindo) according tothe supplier's recommendation, 48 h after tunicamycin treatment.Cytoprotection from ER stress is measured in terms of the percentageincrease in viable cells (relative to control) after ER stress. Theresult for Example 1 of the invention is shown in FIG. 1.

Assessment of Translation Rates in Unstressed Cells (Test 2)

HeLa cells (80,000 cells/ml) were plated in 12-well plates 24 h beforeeach experiment and either untreated or treated with compounds (50 μM)for 0.5, 1, 2.5, 5 and 7.5 h. At the end of each time point, 30.6 μCi/ml³⁵S-methionine (EasyTag, PerkinElmer) was added to the culture mediumfor 10 min at 37° C. Following labelling, cells were washed withice-cold PBS and lysed in 75 μl Laemmli Buffer. Lysates were sonicated,boiled at 95° C. for 5 min and resolved on NuPAGE 4-12% gradient gels.Gels were then stained with Coomassie Brilliant Blue R-250 and analyzedby phosphorimaging.

Assessment of Translation Rates in Stressed Cells (Test 3)

Treatments were performed as for measuring translation in unstressedcells, except that Tunicamycin (2.5 μg/ml) was added together with thecompounds. The result for Example 1 of the invention is shown in FIG. 3.

Immunoprecipitations (Test 4)

HeLa cells (80,000 cells/ml) were plated, the day before the indicatedtreatments, transfected with GFP-PPP1R15A or FLAG-PPP1R15B expressionplasmids using Lipofectamine 2000 (Invitrogen) according tomanufacturer's procedure. Two days following transfection, cells weretreated for 6 h with compounds (50 μM) and then washed in PBS and lysedin IP buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.2% Triton X-100, 10%glycerol, and EDTA-free protease inhibitor cocktail). Lysates wereclarified by centrifugation at 15,000 g for 15 min at 4° C. andpre-cleared on protein G beads for 1 hour at 4° C. Proteins wereimmunoprecipitated with 1.5 μl GFP antibody (JL-8, Clontech, 632380),bound to 20 μl of protein-G-sepharose beads (GE Healthcare, 17-0618-01).The beads were then washed 3 times with cold IP buffer and boiled in 50μl Laemmli Buffer (25 mM Tris-HCl pH 6.8, 1% SDS, 25 mM DTT, 7.5%Glycerol, 0.05% Bromophenol blue). The immunoprecipitated proteincomplexes (17 μl) were separated on 4-12% NuPAGE gradient gels(Invitrogen), transferred to Optitran BA-S 83 reinforced Nitrocellulosemembrane and revealed with GFP and PP1 antibodies (sc-7482, Santa Cruz).

Functional Aequorin Assay for Adrenergic α2A Receptor (Test 5)

CHO-K1 cells coexpressing mitochondrial apoaequorin, Gα16 andrecombinant human Adrenergic α2A receptor grown to mid-log phase inculture media without antibiotics were detached with PBS-EDTA,centrifuged and resuspended in DMEM/HAM's F12 with HEPES, without phenolred+0.1% BSA protease free buffer at a concentration of 1×10⁶ cells/ml.Cells were incubated at room temperature for at least 4 h withcoelenterazine h. On each day of the test, reference agonist (UK14304)was tested to evaluate the performance of the assay and determine EC₅₀.Then, 50 μl of cell suspension was mixed with 50 μl of test agonist in a96-well plate. The resulting emission of light was recorded usingHamamatsu Functional Drug Screening System 6000 luminometer. Tostandardize the emission of recorded light (determination of the “100%signal”) across plate and across different experiments, some of thewells contained 100 μM digitonin or a saturating concentration of AT (20μM).

Dose-response data from test compounds were analysed with XLfit (IDBS)software using nonlinear regression applied to a sigmoidal dose-responsemodel.

The result for Example 1 of the invention is shown in FIG. 4.Advantageously, in contrast to Guanabenz, Example 1: is not consideredto be a potent alpha-2 agonist. This loss in alpha-2 adrenergic activityrenders the compound therapeutically useful in the treatment of thedisorders claimed herein. The absence of alpha-2 adrenergic activitymeans that the compound can be administered at a dosage suitable totreat the disorders claimed herein, but without any significant effecton blood pressure, thereby avoiding the need to co-administer with aknown alpha-2 adrenergic antagonist (an alpha blocker).

Selectivity Assessment

Selectivity was inferred from the results of test 1, 2, 3, and 5:

A selective inhibitor of PPP1R15A should, protect cells from ER stress(Test 1), does not inhibit translation in non-stressed cells (Test 2),prolongs translation attenuation after Tunicamycin (Test 3), andselectively dissociates PPP1R15A-PP1 holophosphatase but notPPP1R15b-PP1 holophosphatase (Test 4).

Results

The results of Tests 1 to 4 for selected compounds of the invention areshown below in Table 1.

TABLE 1 Selectivity Test 1 Test 2 assessment Survival Translation Test 4Selectivity following inhibition Test 3 Dissociation towards ER stressin non- Translation PPP1R15A/PP1 PPP1R15A (increase stressed attenuationor or Ex %) cells after Tm PPP1R15B/PP1^(b) PPP1R15B^(a)  1 180 NOprolonged Dissociation Selectively PPP1R15A/PP1 inhibits BUT PPP1R15A,NOT NOT PPP1R15B/PP1^(b) PPP1R15B  2 40 YES prolonged Inhibits bothPPP1R15A and PPP1R15B  3 80 YES prolonded Preferentially inhibitsPPP1R15A  4 100 YES prolonged Inhibits both PPP1R15A and PPP1R15B  6 120NO prolonged Selectively inhibit PPP1R15A, not B  7 160 YES prolongedDissociation Inhibits both PPP1R15A/PP1 PPP1R15A AND andPPP1R15B/PP1^(b) PPP1R15B  8 100 YES prolonged Inhibits both PPP1R15Aand  9 20 PPP1R15B 13 60-80 15 160 NO prolonged Potentially selective 16140 YES Potentially non selective ^(a)inferred from translationinhibition in stressed/non-stressed cells ^(b)confirms selectivitytowards PPP1R15A-PP1 or lack thereof.Cell-Based Assays:Material and Methods

Cell Culture and Reagents 293T cells were maintained in Dulbecco'smodified Eagle's medium supplemented with 10% fetal bovine serum andtransfected in 6- or 12-well plates by using the calcium phosphatemethod leading usually to 70% transfection efficiency. Routinely, 45,000cells/ml were plated before transfection as described in (Rousseau etal. 2009). Myelin P0S63del-DSred construct was described in (Pennuto etal. 2008), the Huntingtin construct was described in (Rousseau et al.2009), the SOD1A4V constructs described in (Münch. et al. 2011) and theP23H construct is described in (Mendes and Cheetham 2008).

Fluorescence Microscopy

Transfected cells were fixed with 4% paraformaldehyde and labeled withindicated antibodies. Micrographs were taken at 100× magnification on aLeica TCS SP2AOBS confocal microscope or Leica DMRB Fluorescencemicroscope.

Immunoblotting

Routinely, 70% confluent cells from a well of a 12-well plate were lysedin 140 μl of boiling Laemmli buffer (25 mM Tris-HCl, pH 6.8, 1% SDS, 25mM dithiothreitol, 7.5% glycerol, 0.05% bromphenol blue) for immunoblotanalysis. 18 μl of protein extracts were loaded on 2-12% NuPAGE gels andtransferred to Optitran BA-S 83 reinforced nitrocellulose membrane(Whatman and Schleicher & Schuell). Equal loading of protein extractsanalyzed by immunoblot was controlled by Ponceau Red staining andvimentin (data not shown). Membranes were saturated in 5% dried skimmedmilk in phosphate-buffered saline and probed with Htt 2b4 antibody or HAantibody to reveal HA-tagged SOD1. The appropriate secondary antibodycoupled to peroxidase was revealed using the SuperSignal West PicoChemiluminescent kit (Pierce). Chemiluminescent images were acquiredusing the Chemi-Smart 5000 (Vilber-Lourmat) allowing quantitativedetection of chemilumi-nescence. Signals of interest were quantifiedusing ImageJ.

Assay for Charcot Marie Tooth 1B (Test 6)

Deletion of serine 63 from P0 (P0S63del) causes Charcot-Marie-Tooth 1Bneuropathy in humans and a similar demyelinating neuropathy intransgenic mice. The mutant protein misfolds and accumulates in the ER,induces the UPR and fails to be incorporated into myelin (D'Antonio etal., 2009, J Neurosci Res, 87, 3241-9). 293T cells were transfected withlabeled P0S63 del—P0S63del-DSred—and analyzed by confocal microscopy, 48h post-transfection in the presence or absence of compound of formula(I). In accordance with the methodology described in (Pennuto et al.2008), cells with ER-retained P0S63del-DSred were scored. FIG. 4 showsthat in untreated cells, P0S63del accumulates in the ER but Example 1prevents this accumulation. Since accumulation of misfolded P0 causesCMT-1B and having shown that Example 1 reduces accumulation of thedisease-causing protein, the compound of formula (I) should be useful totreat CMT-1B as well as other forms of CMT where the diseasecausing-protein is misfolded and retained in the ER.

Assay for Huntington's Disease and Amyotrophic Lateral Sclerosis (Test7)

We tested for accumulation of mutant huntingtin amino-terminal fragment(Htt48Q) associated with Huntington's disease and SOD1 mutant (A4V),associated with amyotrophic lateral sclerosis.

We used a method previously described in WO/2008/041133. 293T cells weretransfected with plasmids encoding for Htt48 or SOD1^(A4V) and treatedwith Example 1 in DMSO or DMSO alone 4 h post-transfection. SDS lysatescollected 48 h post-transfection were analyzed on a NuPAGE followed byimmunoblot with Huntingtin antibody (2B4) or HA (SOD1). FIG. 5 shows thequantifications of the signal on immunoblots, normalized to untreatedcells. Example 1 reduces accumulation of both proteins. Having shownthat Example 1 reduces accumulation of the proteins causing Huntington'sdisease and Amyotrophic lateral sclerosis, the compound of formula (I)should be useful to treat such diseases as well as otherneurodegenerative diseases caused by accumulation of misfolded proteins.

Assay for Rhodopsin P23H Aggregation (Test 8)

We tested for aggregation of rhodopsin associated for retinitispigmentosa as described (Mendes & Cheetham 2008).

293T cells were transfected with plasmid encoding the P23H mutant ofrhodopsin and treated with Example 1 in DMSO or DMSO alone 4 hpost-transfection. Cells were analyzed by microscopy. FIG. 6 shows thecells with or without aggregates. Example 1 reduces aggregates. Sinceaccumulation of misfolded rhodopsin causes RP and having shown thatExample 1 reduces accumulation of the disease-causing protein, thecompound of formula (I) should be useful to treat Retinitis Pigmentosa.

Examples 1 and 6 are confirmed selective inhibitors of PPP1R15A.

Examples 2,4,7,8 inhibit both PPP1R15A and B.

Various modifications and variations of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the relevant fields are intended to be covered by the presentinvention.

What is claimed is:
 1. A method for treating ALS (amyotrophic lateral sclerosis) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I),

or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein, R₁ is alkyl, Cl, F or Br; R₂ is H or F; R₃ is H or alkyl; R₄ is H or C(O)R₆; R₅ is H; or R₄ and R₅ are linked to form a heterocyclic group which is optionally substituted with one or more R₁₀ groups; R₆ is R₇, OR₇ or NR₈R₉; R₇, R₈ and R₉ are each independently alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocyclyl or aryl, each of which is optionally substituted with one or more R₁₀ groups; each R₁₀ is independently halogen, OH, NO₂, CN, COO-alkyl, aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, CF₃, alkyl or alkoxy; X and Z are each independently CR₁₁, and Y is CR₁₁ or N; and R₁₁ is H or F.
 2. The method according to claim 1 wherein R₁ is Cl, Br, Me, H or F.
 3. The method according to claim 1 wherein R₂ is H.
 4. The method according to claim 1 wherein Y is CR₁₁.
 5. The method according to claim 1 wherein Y is N.
 6. The method according to claim 1 wherein R₃ and R₄ are both H.
 7. The method according to claim 1 wherein R₃ is H and R₄ is C(O)R₆.
 8. The method according to claim 1 wherein R₆ is Me or OMe.
 9. The method according to claim 1 wherein R₄ and R₅ are linked to form a heterocyclic group which is optionally substituted with one or more R₁₀ groups.
 10. The method according to claim 1 wherein said compound of formula (Ia) is,


11. The method according to claim 1 wherein said compound is selected from the group consisting of

or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof.
 12. The method according to claim 11 wherein said compound is Example 1,

or a pharmaceutically acceptable salt thereof.
 13. The method according to claim 11 wherein said compound is Example 15,

or a pharmaceutically acceptable salt thereof.
 14. The method according to claim 11 wherein said compound is Example 16,

or a pharmaceutically acceptable salt thereof.
 15. The method according to claim 1 wherein the ALS is associated with PPP1R15A-PP1.
 16. A method of treating ALS in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of formula (II),

or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein, R₁ alkyl, Cl, F or Br; R₂ is H or F; R₃ is H or alkyl; R₄ is H or C(O)R₆; R₅ is H; or R₄ and R₅ are linked to form a heterocyclic group which is optionally substituted with one or more R₁₀ groups; R₆ is R₇, OR₇ or NR₈R₉; R₇, R₈ and R₉ are each independently alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocyclic, aryl or heteroaryl, each of which is optionally substituted with one or more R₁₀ groups; each R₁₀ is independently halogen, OH, CN, NO₂, COO-alkyl, aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, CF₃, alkyl or alkoxy; X and Z are each independently CR₁₁, and Y is N; and R₁₁ is H or F.
 17. A method of treating ALS in a subject in need thereof, said method comprising administering to the subject, a therapeutically effective amount of a compound of formula (III),

or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein, R₁ is alkyl, Cl, F or Br; R₂ is H or F; R₃ is H or alkyl; R₄ is C(O)R₆; R₅ is H; or R₄ and R₅ are linked to form a heterocyclic group which is optionally substituted with one or more R₁₀ groups; R₆ is R₇, OR₇ or NR₈R₉; R₇, R₈ and R₉ are each independently alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocyclic, aryl or heteroaryl, each of which is optionally substituted with one or more R₁₀ groups; each R₁₀ is independently halogen, OH, CN, NO₂, COO-alkyl, aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, CF₃, alkyl or alkoxy; X and Z are each independently CR₁₁, and Y is CR₁₁ or N; and R₁₁ is H or F.
 18. A method of treating ALS in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the formula (IIIa),

or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein, R₁ is alkyl, Cl, F or Br; R₂ is H or F; R₃ is H or alkyl; and R₁₀ is independently halogen, OH, CN, NO₂, COO-alkyl, aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, CF₃, alkyl or alkoxy.
 19. A method of treating ALS in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of formula (IV),

or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof wherein, R₁ is alkyl or Br; R₂ is H; R₃ is H or alkyl; R₄ is H or C(O)R₆; R₅ is H; or R₄ and R₅ are linked to form a heterocyclic group which is optionally substituted with one or more R₁₀ groups; R₆ is OR₇ or NR₈R₉; R₇, R₈ and R₉ are each independently alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocyclyl or aryl, each of which is optionally substituted with one or more R₁₀ groups; each R₁₀ is independently halogen, OH, CN, NO₂, COO-alkyl, aralkyl, SO₂-alkyl, SO₂-aryl, COOH, CO-alkyl, CO-aryl, NH₂, NH-alkyl, N(alkyl)₂, CF₃, alkyl or alkoxy; X and Z are each CH and Y is CR₁₁; and R₁₁ is H or F.
 20. A method of treating ALS in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of

or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof.
 21. The method according to claim 1 wherein the compound is administered by oral route.
 22. The method according to claim 1 wherein the compound is the compound of Example 1,

or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof.
 23. The method according to claim 1 further comprising administering one or more other active agents.
 24. The method according to claim 1 wherein R₁ is Cl. 